The present invention relates to methods and apparatus for delivery of pharmaceuticals to target tissues in situ, in vivo, ex vivo, or in vitro.
Advances in recombinant-DNA technology have made introduction of therapeutic genes into somatic cells possible (Anderson, Nature 357:455-457, 1992). In recent years, several clinical trials involving human gene therapy have been accepted by regulatory agencies. The initial human gene therapy clinical trials aimed at treating both inherited diseases (such as severe combined immunodeficiency caused by lack of adenosine deaminase in peripheral T-lymphocytes, cystic fibrosis, and familial hypercholesterolemia), as well as noninherited disease such as cancer (Wolfe, Curr. Opinion in Pediatr. 6: 213-219, 1994; Sanda et al., J. Urology 44:617-624, 1994; O""Malley et al., Arch. Otolaryngol. Head Neck Surgery 119:1191-1197, 1993; Engelhardt et al., Nature Genetics 4: 27-34, 1993; Lemarchand et al., PNAS (USA) 89: 6482-6486, 1992; Jaffe et al., Nature Genetics 1:372-378, 1992).
The development of suitable, safe, and effective gene transfer systems is a major goal of research in gene therapy. Thus far, viruses have been extensively used as vectors for gene therapy. (See, for example, Pilewshi et al., Am. J. Physiol. 1995;268(4 pt 1):L657-665; Prince, Pathology 1998;30(4):335-347). For example, retroviruses have been widely used, but they can only target actively dividing cells, and do not readily accommodate large DNA inserts. Adeno-associated viruses are also limited in the ability to accommodate large inserts, yet replication defective adenoviruses have been used successfully to transfer of a variety of genes into cells in culture and in vivo. Adenoviruses can accommodate larger inserts than retroviruses, but extra-chromosomal expression usually lasts only for a few weeks. Herpes viruses have been exploited for specific gene transfer trials into the central nervous system. Herpes viruses can carry large foreign DNA inserts, and may remain latent for long periods of time.
In spite of the availability of replication defective viruses, concerns about the safety and efficiency of such viral vectors have generated interest in the development of non-viral gene transfer systems such as liposome-DNA complexes and receptor mediated endocytosis (Felgner P. L. et al., PNAS (USA) 84: 7413-7417, 1987; Hyde Nature 362: 250-255, 1993; Nu G. Y. J. Biol. Chem. 266: 14338, 1991).
A major hurdle for effective gene therapy is the development of methods for targeting the gene transfer to appropriate target cells and tissues. Ex vivo gene transfer into explanted cultured cells and implantation of the treated cells has been used for the treatment of hematopoietic tissues (U.S. Pat. No. 5,399,346, hereby incorporated by reference), and bronchial epithelial cells in animal model. (Engelhardt et al., Nat Genet 1993;4:27-34) Also, direct injection into brain and lung tumors (Cusack et al., Cancer Gene Ther 1996; 3(4):245-249), intravenous or intra-arterial administration (Schachtner et al., Circ Res 1995; 76:701-708), inhalation (Katkin et al., Hum Gene Ther 1995; 6:985-995), and topical application (Pilewshi et al., Am J Physiol 1995;268(4 pt 1):L657-665) have been used. Major drawbacks to all of these methods are that the transduction is not highly selective, significant amounts of the therapeutic gene containing vector may be needed, and efficiency of the gene transfer is severely limited by the constraints of vector concentration, time of exposure to the target, and effectiveness of the gene transfer vector.
Much research is being conducted to enhance transgene expression in target cells. Gene transfer efficiency has been reported to improve by pretreatment with host barrier properties modificating agents (e.g polidocanol), before vector administration. (Parsons et al., Hum Gen Ther Dec. 10, 1998; 9(18):2661-72). Modification of the host""s immune system may enhance the transgene expression in viral mediated gene transfer. (Ghia et al., Transplantation Dec. 15, 1998; 66(11):1545-51) Another method reported to enhance gene transfer efficacy is prolonging the incubation time with the vector and the target cells. (Zabner et al., J. Virol. 1996; 70;6994-7003)
One area of active research is gene therapy into mammalian kidneys, but the results have been disappointing because of poor gene transfer efficiency (Woolf et al., Kidney Int. 43: Suppl. 39: S116-S119, 1993). Moullier et al. showed some adenovirus-mediated transfer of lacZ gene into rat tubular, but not glomerular cells, following a combination of virus infusion into the renal artery and retrograde infusion into the vector (Kidney Int. 45: 1220-1225, 1994). Simple infusion of soluble virus does not appear to be an efficient transfer system. Better results were obtained by Tomita et al., (Biochem. Biophys. Res. Commun. 186: 129-134, 1992), who infused a complex of Sendai virus and liposomes into the rat renal artery in vivo, resulting in marker gene expression in about 15% of the glomerular cells.
Alport syndrome is an inherited kidney disease characterized by progressive hematuria, development of renal failure and frequently also hearing loss (Atkin C L and Gregory M C: Alport syndrome; IN: Schrier W W, Gottschalk C W, eds. Diseases of Kidney, Little Brown, Boston 1993 pp 571-592; Tryggvason K, Heikkilxc3xa4 P: Alport syndrome. In: Jamison L, ed. Principles of molecular medicine, Humana Press Inc.
Totowa N.J. USA 1998 pp 665-668). The only available treatment is hemodialysis and/or kidney transplantation. The underlying cause of the disease is defective structure of the type IV collagen meshwork of the glomerular basement membrane (GBM). This typically results in abnormal thinning and thickening and a basket-weave-like pattern of the GBM. The disease affects about 1:5,000 males (Atkin C L and Gregory M C: Alport syndrome; IN: Schrier W W, Gottschalk C W, eds. Diseases of Kidney, Little Brown, Boston 1993 pp 571-592). About 85% of the cases are caused by mutations in the X chromosomal gene for the type IV collagen xcex15 chain (Barker, D., Hostikka, S. L., Zhou, J., Chow, L. T., Oliphant, A. R., Gerken, S. C., Gregory, M. C., Skolnick, M. H., Atkin, C. L. and Tryggvason, K.: Identification of mutations in the COL4A5 collagen gene Alport syndrome. Science 248, 1226-1227, 1990, Hostikka S L, Eddy R L, Byers M G, Hxc3x6yhtyxc3xa4 M. Shows T B. Tryggvason K Identification of a distinct type IV collagen a chain with restricted kidney distribution and assignment of its gene to the locus of X chromosome-linked Alport syndrome. Proc Natl. Acad Sci USA 1990:87:1606-1610, Tryggvason, K Mutations in type IV collagen genes and Alport phenotypes. In: Molecular Pathology and Genetics of Alport Syndrome (Ed. Karl Tryggvason), Karger, Basel, Vol. 117, pp. 154-171, 1996). The less frequent autosomal forms are caused by mutations in the type IV collagen xcex13 or xcex14 chain genes located on chromosome 2 (Mochizuki T, Lemmink H H, Mariyama M, Antignac C, Gubler M-C, Pirson Y, Verellen-Dumoulin C, Chan B, Schxc3x6der C H, Smeets H J, Reeders S T: Identification of mutations in thexe2x80x943(IV) and 4(IV) collagen genes in autosomal recessive Alport syndrome. Nature Genet 1994;8: 77-81, Lemmink K K, Mochizuki T, van den Heuvel L P W J, Schrxc3x6der C H, Barrientos A, Monnens L A H, van Oost B A, Brunner H G, Reeders S T, Smeets J M mutations in the type IV collagen xcex13 (COL4A3) gene in autosomal recessive Alport syndrome. Hum Mol Genet 1994;3:1269-1273.
Type IV collagen is a basement membrane specific collagen type which is the main structural component of these extracellular structures (Hudson B G. Reeders S T. Tryggvason K Type IV collagen: Structure, gene organization and role in human diseases. Molecular basis of goodpasture and Alport syndromes and diffuse leiomyomatosis. J Biol chem. 1993:268:26033-26036). Similarly to other collagens type IV collagen is a triple-helical protein consisting of three xcex1 chains. The collagen xcex1 chains have (Gly-Xaa-Yaa)n repeats, glycine being the only amino acid small enough to fit into the center of the triple helix. The type IV collagen xcex1 chains have many interruptions in the Gly-Xaa-Yaa repeat which allows the formation of flexible kinks in the triple-helical molecules. In addition to the collagenous domain, the type IV collagen molecules have noncollagenous globular NCl domain at the carboxyl end, and the aminoterminal 7S domain. Six genetically distinct type IV collagen xcex1 chains have been described. The xcex11(IV) and xcex12(IV) chains are ubiquitous and are present in triple-helical molecules in a 2:1 ratio (Kxc3xchn K Basement membrane-type collagen. Matrix Biol 1994:14:439-455). The other a chains vary in their more restricts tissue distribution. The current understanding type IV collagen synthesis in the renal glomerulus is illustrated in FIG. 5. In the GBM, xcex11(IV) and xcex12(IV) are prominent during embryonic development (FIG. 5), but after birth these are replaced by xcex13(IV), xcex14(IV) and xcex15(IV) chains (Miner J H, Sanes J R: Collagen IV (xcex13, xcex14, and xcex15 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches. J Cell Biol 1994p;127:879-891) that, because of high cysteine content (Leinonen A. Mariyama M. Mochizuki T. Tryggvason K. Reeders S T Complete primary structure of the human type IV collagen xcex14(IV) chain; comparison with structure and expression of the other xcex1(IV) chains. J Biol Chem. 1994;269:26172-26177) are thought to be necessary for forming a stronger, more cross-linked network of triplehelical molecules composed of xcex13(IV), xcex14(IV) and xcex15(IV) chains in a 1:1:1 ratio (Gunwar S. Ballester F. Noelken M E. Sado Y. Ninomiya Y. Hudson B G: Glomerular basement membrane; identification of a novel disulfide cross-linked network of xcex13, xcex14 and xcex15 chain of type IV collagen and its implication for the pathogenesis of Alport syndrome. J Biol Chem. 1998;273:8767-8775)(see FIG. 5). In X-linked Alport syndrome caused by a mutation in the xcex15(IV) chain gene, the xcex13(IV) and xcex14(IV) chains are usually absent from the GBM, even though their genes reside on chromosome 2 (Nakanishi K. Yoshikawa N, Iijima K, Kitagawa K, Nakamura H, Ito H, Yoshioka K, Kagawa M, Sado Y: Immunohistochemical study of xcex11-xcex15 chains of type IV collagen in hereditary nephritis. Kidney Int. 1994;46:1413-1412). This is presumably due to intracellular degradation of the chains in the absence of xcex15(IV) that is essential for the xcex13:xcex14:xcex15 trimer. Instead, the GBM contains embryonic type of collagen IV molecules consisting of al and xcex12 chains (FIG. 5). However, since these apparently do not provide sufficient mechanical strength to the GBM and sufficient resistance to proteolysis (Kalluri R. Shield III C F, Todd P. Hudson B G Neilson E G Isoform switching of type collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis. J clin Invest 1997;99:2470-2478), the consequence is deterioration of the structure and development of Alport syndrome.
Alport syndrome is an attractive candidate disease for gene therapy due to its high kidney specificity and because the isolated blood circulation of the kidneys makes them a good target for organ specific gene transfer. The principle of gene therapy of Alport syndrome is depicted in FIG. 5. This requires transfer of the appropriate type IV collagen a chain gene to the endothelial an epithelial cells of the glomerulus, expression of the protein and intracellular assembly of the exogenous recombinant chain into triple-helical molecules together with the endogenous or xcex13, xcex14 or xcex15 chains, and finally, secretion of the protein and incorporation of the protein into the GBM type IV collagen network (see FIG. 5)(Tryggvason K, Heikkilxc3xa4 P, Pettersson E, Tibell A, Thomer P. Can Alport syndrome be treated by gene therapy? Kidney Int 1997;51:1493-1499). For the development of gene therapy of Alport syndrome, we have previously developed an organ perfusion system for adenovirus-mediated gene transfer into renal glomeruli in vivo (Heikkilxc3xa4 P. Parpala T. Lukkarinen O. Weber M. Tryggvason K. Adenovirus-mediated gene transfer into kidney glomeruli using an ex vivo and in vivo kidney perfusion systemxe2x80x94first steps towards gene therapy of Alport syndrome. Gene Therapy 1996;3:21-27). Using this procedure we obtained a transfer efficiency up to 85% of pig glomeruli using an adenovirus containing the xcex2-galactosidase reporter gene. Surprisingly, the kidney perfusion method only revealed efficient transfer to glomerular cells, while cells in other regions of the kidney did not take up significant amounts of the virus.
Another active research area is gene therapy into the lung. To date, most gene therapy approaches to both inherited and acquired lung diseases have involved viral or liposome mediated gene delivery via the airway, which provides direct access to lung epithelia. (Griesenbach et al., Gene Ther 1998;5:181-188) At least one drawback of the aerosol delivery, especially in advanced cystic fibrosis (CF), is that the infected mucus layer in bronchioles may impair access to the cell surface. So far, intravascular infusions of the vectors have yielded quite inefficient gene transduction.
There are a variety of diseases that are candidates for somatic lung directed gene therapy, including CF and xcex11-antitrypsin deficiency, which are the most common inherited diseases having serious pulmonary manifestations. The first reports of in vitro correction of the CF chloride channel defect came in 1990 (Drumm et al., Cell 1990; 62:1227-1233) and in vivo CF gene expression could be established in the airways of mice in 1992. (Rosenfeld et al., Cell 1992;68:143-155) Another candidate for lung directed gene therapy is the surfactant protein B deficiency, an autosomal recessive pulmonary disease, which manifests in neonates and leads to lethal respiratory failure within the first year of life. Gene therapy is also being considered for the treatment of inflammatory and infectious diseases and of cancer of the lung. (Dubinett et al., Hematol Oncol Clin North Am 1998;12(3):569-94)
It would be extremely beneficial to the medical arts to have apparatuses and methods for the efficient administration of gene therapy to target cells and tissues that overcome the limitations inherent to the various gene transfer vector.
In accordance with an aspect of the present invention, there are provided methods for the administration of pharmaceuticals to targets for functional use. The term xe2x80x9cpharmaceutical,xe2x80x9d as used herein, includes chemical drugs, protein drugs, nucleic acid drugs, combination chemical/protein/nucleic acid drugs, and gene therapy vectors.
The term xe2x80x9cfunctional use,xe2x80x9d as used herein, includes therapeutic treatment, prophylaxis, and/or production of recombinant proteins in vivo. The term xe2x80x9cfunctional usexe2x80x9d also includes the disruption of endogenous gene expression including the use of antisense, triplex forming, catalytic and otherwise disruptive pharmaceuticals. The term xe2x80x9cfunctional usexe2x80x9d also includes the expression of recombinant proteins in target tissues, whether of endogenous or exogenous origin. The term xe2x80x9ctarget,xe2x80x9d as used herein, includes cells, tissues and/or organs.
The term xe2x80x9cgene therapy vectorxe2x80x9d is meant to include nucleic acid constructs which are single, double or triplex stranded, linear or circular, that are expressible or nonexpressible constructs which can either encode for and express a functional protein, or fragment thereof, or interfere with the normal expression of a target gene, gene transfer and/or expression vectors.
The administration of pharmaceuticals may take place where the target is in situ in a living subject. The administration may also take place wherein the target is first removed from a subject, manipulated ex vivo, and returned to the original or, alternatively, to a second recipient subject. In a preferred embodiment, the target is situated such that the circulation of the blood supply into and out of the target is relatively isolated. In a most preferred embodiment, the blood circulation into and out of the target is mostly via a single, or readily identified entering arteries and exiting veins. There are of course certain amounts of limited leakage due to small blood and lymphatic vessels.
The methods of the instant invention allow for a prolonged period of administration of pharmaceuticals to a target by way of re-circulating a pharmaceutical containing solution through the target such that a perfusion effect occurs. The methods of the instant invention allow for prolonged administration because of the unique use of the perfusion method and the oxygenation of the pharmaceutical containing solution. In one embodiment, the perfusion apparatus and target forms a closed system whereby the pharmaceuticals are administered at a starting concentration and not adjusted during the time course of treatment. In another embodiment, the pharmaceutical concentration is periodically adjusted so as to maintain or otherwise alter the concentration of pharmaceutical in the solution, or additional pharmaceuticals are added. In a preferred embodiment, the solution does not require replenishment during the course of treatment. In another embodiment, the solution volume can be replenished as leakage or other forms of loss occur during the course of treatment. (The term xe2x80x9csolution,xe2x80x9d as used herein refers to the medium in which the pharmaceutical is suspended, dissolved or otherwise maintained for delivery to the target, aka. the perfusate, and includes blood, serum, plasma, saline, and/or buffered solutions.) In a preferred embodiment, 350 ml of perfusate contains red blood cells (around 17% of hemocrit value), and can include about 25,000 IU heparin, about 20,000 IU penicillin and about 20,000 xcexcg streptomycin in Krebs-Ringer solution in addition to the pharmaceutical.
The instant invention also provides methods for delivering viral vector gene therapy pharmaceuticals to a mammalian target tissue comprising contacting the mammalian target tissue with the viral vector gene therapy pharmaceutical in a re-circulating, oxygenated perfusate solution, where said solution is held at about 37xc2x0 C., such that there is effective delivery of the viral vector gene therapy pharmaceutical. In a preferred embodiment, the mammalian target tissue is selected from kidney, liver, mammary glands, spleen, and lung.
In another embodiment, the present invention provides methods for the extended delivery of a pharmaceutical to mammalian kidney tissue comprising contacting the mammalian kidney tissue with the pharmaceutical in a re-circulating, oxygenated perfusate.
The instant invention further provides improved methods for gene therapy of kidney disorders, comprising contacting the kidney of a patient with a kidney disorder with an amount effective for treatment of the disorder of a gene therapy pharmaceutical for treatment of the disorder, wherein the improvement comprises contacting the patient""s kidney with the viral vector gene therapy pharmaceutical in a re-circulating, oxygenated perfusate solution.
In a more particular embodiment, the invention provides for treatment of Alport syndrome in a patient by gene therapy, comprising contacting the kidney of a patient with Alport syndrome with recombinant xcex15(IV) chain, obtained from human type (IV) collagen xcex15 cDNA, using a re-circulating, oxygenated perfusate solution.
The instant invention also provides for a perfusion apparatus functionally connected by a perfusate transfer system comprising, (a) a reservoir for perfusate, (b) means for propelling the perfusate through the apparatus, (c) means for oxygenation of the perfusate, (d) means for connecting the apparatus to and from the target.
The reservoir for the perfusate can be any container that can be sterilized and used to collect perfusate from the target. The reservoir is connected to the means for transporting the perfusate through the system by means of tubing. While perfusion may occur at room temperature of 20xc2x0 C., in a preferred embodiment, the perfusion occurs at 37xc2x0 C. Thus, in practice, the perfusate reservoir can be maintained at any desired temperature via, for example, a water bath.
In an embodiment where the means for propelling the perfusate is a peristaltic pump, the tubing is preferably silicone or other such suitable pliable tubing. Where the means for propelling the perfusate is a peristaltic pump, no contact is made between the perfusate and any part of the pump directly. In the case where a pump with, for example an impeller blade is used, then the perfusate comes into direct contact with a part of the pump. In the usual configuration using a peristaltic pump, the tubing from the reservoir passes through the pump and connects with the means for oxygenating the perfusate.
The means for oxygenating the perfusate can be any form of artificial lung, or aeration device such that the perfusate is oxygenated without overt agitation and subsequent frothing. In one embodiment the means for oxygenating the perfusate is a membrane lung which consists of a length of semi-permeable tubing packed into a gas chamber into which is circulated oxygen rich gas, for oxygenating the perfusate as it pass through the length of tubing. In a preferred embodiment, the membrane lung contains about 8 meters of silicon tubing of approximately 1.47 mm inside diameter, and the gas circulated in the chamber is carbogen gas (comprised of 95% oxygen, 5% carbon dioxide).
In general, the target is cannulated and connected to tubing connecting from the means for oxygenating the perfusate, and leading to the perfusate reservoir. In one configuration, the perfusate is pumped from a reservoir, through a means for oxygenating the perfusate, into the target, through the target, and back into the reservoir. The location of the pumping means in relation to the other components can be varied. The number of each component can also be varied.
Thus the instant invention provides for a method of administering a pharmaceutical to a target whereby the target is mostly isolated and continuously perfused with a perfusate containing the pharmaceutical, and said perfusate is recirculated and oxygenated. The instant invention provides for an apparatus for the administration of pharmaceuticals to a target comprising a perfusate reservoir, means for pumping the perfusate, means for oxygenating the perfusate, and means for connecting the components to one another, and with the target. In a preferred embodiment the re-circulating perfusion apparatus comprises a perfusate reservoir receiving efflux perfusate from the target, connected with silicone tubing passing via a peristaltic pump to a membrane lung, said membrane lung comprising about 8 m of approximately 1.47 mm inner diameter silicone tubing immersed in a circulating gas chamber filled with carboxygen gas, connected by tubing and a catheter to a target.